The present disclosure relates to quantitative fluorescence imaging and more specifically to an autofocus system and method using angular illumination.
A recent improvement in fluorescence imaging is a bi-telecentric, wide-field fluorescence scanner that allows for accurate quantification measurements with focus-independent pass-to-pass registration. U.S. patent application Ser. No. 14/312,409, filed Jun. 23, 2014, titled “Telecentric, Wide-Field Fluorescence Scanning Systems and Methods,” which is hereby incorporated by reference for all purposes, discusses features of a bi-telecentric, wide-field fluorescence scanning system. One feature of such a design is angular illumination (excitation), which has the benefit of reduced optical background and therefore higher sensitivity. A down side of angular illumination, however, is that as the height of the sample changes, the location of the imaged line on the sensor changes as well. Therefore, in order to fully take advantage of this angular illumination feature, it is important to track where the excitation light hits the sample as its height changes.
Another feature that the bi-telecentric scanner can use is a differential scan imaging technique to achieve high image performance by subtracting background signal from a non-illuminated area from the signal detected from an illuminated area. U.S. patent application Ser. No. 13/084,371 filed Apr. 11, 2011, titled “Differential Scan Imaging Systems and Methods,” which is hereby incorporated by reference for all purposes, discusses useful differential scan imaging techniques. When using such techniques, combined with angular illumination, it is desirable that the z-height of the fluorescence sample be constant relative to the scanner system. If the sample height changes, the detected signal from the illuminated area ‘walks off’ across the detection array and therefore the amount of signal measured is not accurate. Therefore, with a given stationary detector array in place, it is important to correct for sample height changes before actual imaging data is collected.
Olsen et al. (U.S. Pat. Nos. 7,518,652, 7,646,495, and 7,893,988, which are each hereby incorporated by reference) devised ways to focus a line scan camera prior to and during the capture of imagery data from a specimen on a microscope slide. The approach taken consists of computing focus information prior to scanning the slide. This focus information is taken in a point focus or ribbon-focus procedure. In the point focus case, the line scan camera system first positions the slide at a desired measurement location, moves the objective lens through a predefined set of height values, and acquires imagery data at each height and then determines the height (z-axis setting) of maximum contrast, which in turn is established as the optimal focus height. With the ribbon-focus procedure, the objective is continuously moved in a sinusoidal fashion as the slide is in scanning motion. Imagery data are analyzed and heights of maximum contrast determine the best focus z-heights. The two procedures differ in how the vertical motion of the objective lens is synchronized with the horizontal motion of the slide during image acquisition. The first method, which can be described as a ‘stop-and-go’ method, is slow as there is quite a bit of overhead time as a result of the stop-and-go process. The ribbon-focus method is much faster, but still takes more than 1 min for a 15 mm×15 mm scan area.
Olsen's method was devised for microscopic imaging where illumination light comes through the microscope objective, i.e. co-axial or non-angular relative to the imaging path. This means that as the vertical distance between the objective and sample changes (z-axis), the location on the imaging sensor, in the x-y plane, does not change—just image contrast changes. In this microscope configuration, it does make sense to adjust the objective position to different height locations and finding the best position with the image having the highest contrast. However, with angular illumination, the x-y location on the imaging sensor does change as the focus changes. This means that additional steps to find the x-y location would be needed before Olsen's ‘stop-and-go’ process of finding best focus can be implemented. An even more elaborate set of steps would be needed for the strip-focus method. This adds complication and slows the process even further.
Furthermore, Olsen's technique has no provisions for the detected signal walking off on the detector as a result of z-height changes of the sample. Therefore, the idea of taking images at different heights does not work because at many z heights there would be no signal to detect (walked off the detector) and thus any contrast-based scheme would fail to detect where the best focus is. It is therefore necessary to bring the sample height near the nominal height that gives best focus first so it can be measured correctly, e.g., using Differential Scan Imaging techniques.
Therefore, there is still a need for a more robust, quantitative, fast macroscopic fluorescence imager that does not have the limitations of angular dependence on where in the field the light originates from. Furthermore, there is still a need to accurately maintain the relative locations of the origins of fluorescence light on the sample so that multi-pass images are aligned accurately and thus eliminate the focus dependent positional shifting present in current macroscopic wide-field imagers.